Ischemic heart disease (IHD) is the leading cause of mortality in this nation. Studies have shown that more premenopausal women are protected from IHD than age-matched men. However, the mechanisms underlying the gender difference are not clear. Scientific data show that the endogenous female hormone, 17-betaestradiol (E2) may be responsible for protecting heart muscle cells (cardiomyocytes). Our goal is to determine how E2 protects cardiomyocytes under ischemic stress. We hypothesize that estrogen signals to several important regulators of cell fate in ischemic stress - cardiac p38 mitogen-activated protein kinases (p38 MAPKs) and p53, a well-known mediator of apoptosis - to protect the cardiomyocyte. We will investigate how E2 mediates the novel relationship between cardiac p38 MAPKs and p53 in mitochondria-driven cell death, the dominant pathway of cardiac apoptosis. In Specific Aim 1, we will establish the role of E2 in cardiac p38 MAPK regulation. The p38 MAPK subtypes, p38 and p38, possess distinct independent functions, with the former participating in apoptosis and the latter in preservation of cellular structure. We will subject cultured rat cardiomyocytes to simulated ischemia-reperfusion injury (I/R) and [A] examine the effect of E2 on each p38 isoform; [B] correlate the E2-specific effects on each kinase to mitochondria-centered apoptosis; and [C] identify the target(s) of p38 and p38 in the mitochondria. We will correlate data from cells to an in-vivo model of I/R in female ovariectomized rats with or without E2 supplement and examine p38, p38, cardiac apoptosis in situ, and cardiac function by echocardiography. In Specific Aim 2, we will investigate how E2 mediates p38 MAPKs - p53 interaction. p53 is a substrate of pro-apoptotic p38 in different cell types. We will [A] establish that p53 is phosphorylated by the p38 in cardiomyocytes; and [B] detail the nature of the posttranslational modification with respect to p38-mediated cell death. We will also [A] investigate if p38 phosphorylates p53, and if so, what biological significance such a relationship bears in I/R-triggered apoptosis; [B] explore a possibility of a feedback mechanism between p53 and p38; [C] determine how E2 modulates the dynamic between p53 and p38; and [D] translate and confirm findings in a whole animal model of I/R injury by detailing p53-specific changes in cardiac p38 expression and activation at the organ level. In Specific Aim 3, we will define the role of estrogen receptor (ER) subtypes in cardiomyocyte protection. We suspect that both ER subtypes (ER and ER) are necessary for full cytoprotection from mitochondria-centered apoptosis. We will [A] delineate how each receptor signals to p38 and subsequent regulation of oxidative stress in mitochondria [B] identify and characterize endogenous ER / heterodimers in cardiomyocyte mitochondria, and [C] express a fusion ER / protein in ER-null cells and compare with ER homodimers in its ability to regulate apoptosis, mitochondrial p53 and p38 function.